Our laboratory is primarily involved in studies dealing with the regulation of steroid metabolism in monolayer cultures of ACTH responsive mouse adrenal cortex tumor cells, and the elucidation of the mechanism of action of ACTH and other activators of steroid synthesis. They provide a sensitive and stable model system for investigating the initial events in the activation of steroidogenesis as well as long-term effects of ACTH on enzyme systems essential to the differentiated function of the cell. This proposal involves three areas of work: 1) The elucidation of mechanisms by which the levels of specific mitochondrial proteins are altered following ACTH stimulation. We shall identify mitochondrial proteins which are increased or decreased as a result of stimulation with ACTH and correlate these increases with alterations in mitochondrial enzyme activity. We shall determine the genetic origin of these proteins and try to determine the mechanism by which they are increased, e.g., increased gene dosage, transcription, translation, etc. The effects of other stimulators of steroidogenesis, e.g., cyclic-AMP, adenosine, cholera toxin and VIP will also be assessed. 2) As a followup to our studies of the kinetics of ACTH and cholera toxin stimulation of cyclic-AMP and steroid synthesis we propose to characterize the effects of the active sub-unit of the toxin in both intact cells and isolated membranes and the relationship of the structure of cholera toxin to its activity. This will include isolation of sub-units, identification of peptide fragments containing active site and the effect of certain fragments of the toxin on toxin binding to the intact cell. Chemical analysis of the cell surface membrane receptor for both the binding and active sub-unit of the toxin will also be investigated. Similar investigations will be carried out with Vasoactive Intestinal Peptide (VIP), a peptide which stimulates steroidogenesis but has little effect on cyclic-AMP metabolism. 3) We shall continue our studies of the effects of ACTH and other activators of steroidogenesis on cell ultrastructure with particular emphasis on mitochondria, microfilaments and microtubules.